CN104212850A - Method for preparing 1-cyancyclohexylacetic acid by using nitrilase engineering bacterium - Google Patents
Method for preparing 1-cyancyclohexylacetic acid by using nitrilase engineering bacterium Download PDFInfo
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Abstract
The invention discloses a method for preparing 1-cyancyclohexylacetic acid by using a nitrilase engineering bacterium. The method comprises the following steps: centrifuging a culture liquid obtained after fermenting culture of an engineering bacterium containing recombinant nitrilase gene, carrying out a conversion reaction at 25-70DEG C with wet thallus as a catalyst, 1-cyancyclohexyl acetonitrile as a substrate and a buffer solution with the pH value of 3.0-10.0 as a reaction medium to obtain a mixed solution containing 1-cyancyclohexylacetic acid, and separating and purifying the mixed solution to obtain 1-cyancyclohexylacetic acid. The engineering bacterium containing recombinant nitrilase gene is constructed by gene encoding a nucleotide sequence represented by SEQ ID NO.2. The engineering bacterium containing recombinant nitrilase gene has the advantages of good regioselectivity, good catalytic vitality, and environmental protection in the production process as a catalyst.
Description
(1) technical field
The present invention relates to a kind of preparation method of gabapentin key intermediate 1-cyanocyclohexanoic guanidine-acetic acid, a kind of particularly method utilizing nitrilase engineering bacteria to prepare 1-cyanocyclohexanoic guanidine-acetic acid.
(2) background technology
1-cyanocyclohexanoic guanidine-acetic acid is the key intermediate of synthesis gabapentin, and main chemical method is produced at present, severe reaction conditions, and a large amount of alkali liquor absorption of waste gas palpus of generation, step is more, and yield is lower.
Nitrilase can realize the organic carboxyl acid of a step regioselective hydrolyis synthesis correspondence of organonitrile compound.Realize cyan-hydrolysis reaction conditions by nitrilase gentle, reaction efficiency is high, and has higher regioselectivity and stereoselectivity.In recent years, utilizing nitrilase to carry out cyan-hydrolysis synthesis organic carboxyl acid has become the focus of research, lot of documents, patent reports and utilizes the application of the regioselectivity of nitrilase in organic carboxyl acid synthesis.
The people such as Zhu report nitrilase bll6402 (the Dunming Zhu come from bacterial strain Bradyrhizobium japonicum USDA110, Adv.Synth.Catal.2007,349,1667-1670), find that this nitrilase can catalysis various pairs of nitriles, to the two nitrile (comprising 1-cyanocyclohexanoic base acetonitrile and analogue thereof) of part, there is higher regioselectivity.
The people such as Sophie Bergeron report a strain nitrilase BD9570 (from Diversa) (Sophie Bergeron, Organic Process Research & Development 2006,10,661-665), this nitrilase can synthesize atorvastatin side chain crucial chiral intermediate (R)-4-cyano-3-hydroxy butyric acid by catalysis 3-HGN, has higher regioselectivity and stereoselectivity.
The people such as Zhiyi Xie report nitrilase AtNit1 (the Zhiyi Xie that a strain comes from Arabidopsis thaliana, Journal of Molecular Catalysis B:Enzymatic, 2006,41,75 – 80), this nitrilase can synthesize lyrica chiral intermediate (3S)-3-cyano group-5-methylhexanoic acid by catalyzing iso-butane base succinonitrile, has very high regioselectivity and stereoselectivity.
The people such as John E.Gavagan report a strain and derive from Acidovorax facilis 72W nitrilase (John E.Gavagan, J.Org.Chem., 1998,63,4792-4801), this aliphatic nitrilase has good regioselectivity for α, ω-bis-nitrile.Patent US2005009157 A1 also reports this nitrilase can catalysis 1-cyanocyclohexanoic base acetonitrile synthesis gabapentin intermediate 1-cyanocyclohexanoic guanidine-acetic acid.
Patent US2005009157 A1 there was reported commercialization nitrilase NIT-104, NIT-105, NIT-106 (from Biocatalytics Inc.) and for 1-cyanocyclohexanoic base acetonitrile, there is regional choice Sexual potency from the nitrilase of bacterial strain Bacillus sphaericus.
The people such as Carmela Bengis-Garber report the nitrilase in bacterial strain Rhodococcus rhodochrous N.C.I.B.11216 and have regioselectivity for the catalysis of α, ω-bis-nitrile.And the carbon chain lengths of the regioselectivity of this bacterial strain and substrate α, ω-bis-nitrile and inductor used when cultivating relevant.(Carmela?Bengis-Garber,Appl?Microbiol?Biotechnol(1989)32:11-16)
The people such as Chandrani Mukherjee report nitrilase (the Chandrani Mukherjee that a strain comes from Synechocystis sp.Strain PCC6803, Eur.J.Org.Chem.2006,5238 – 5242), the various α of this nitrilase catalysis, ω-bis-nitrile, the carbon chain lengths of its regioselectivity and α, ω-bis-nitrile has very large relation.Along with the increase of carbon chain lengths, the regioselectivity of this nitrilase declines gradually.
Up to now, about nitrilase report 1-cyanocyclohexanoic base acetonitrile to regioselective catalytic vigor is less, main from Acidovorax facilis 72W, Bradyrhizobium japonicum USDA110, Bacillus sphaericus and commercialization nitrilase NIT-104, NIT-105, NIT-106.But there is the problem that catalytic activity is lower and catalyst levels is larger in these nitrilase catalysis 1-cyanocyclohexanoic guanidine-acetic acid.So screening New biocatalyst, develop efficient gabapentin key intermediate enzymatic clarification technique, the gabapentin new synthetic process setting up green economy is significant.
(3) summary of the invention
The object of the invention is that solving traditional chemical method hydrolysis 1-cyanocyclohexanoic base acetonitrile is in the technique of 1-cyanocyclohexanoic guanidine-acetic acid, and severe reaction conditions, need a large amount of organic solvents in reaction, cost is higher, and yield is lower, the problem that environmental pollution is comparatively serious.
The invention provides a kind of regioselectivity nitrilase being hydrolyzed 1-cyanocyclohexanoic base acetonitrile, this enzyme has good regioselectivity and catalysis activity when the above-mentioned reaction of catalysis, and production process is environmentally friendly.
The technical solution used in the present invention is:
The invention provides a kind of method utilizing nitrilase engineering bacteria to prepare 1-cyanocyclohexanoic guanidine-acetic acid, described method is: by the medium centrifugal of engineering bacteria after fermentation culture containing restructuring nitrilase gene, using wet thallus as catalyzer, with 1-cyanocyclohexanoic base acetonitrile for substrate, take pH value as the damping fluid of 3.0 ~ 10.0 be reaction medium (preferable ph is the phosphoric acid buffer of 7), conversion reaction is carried out at 25 ~ 70 DEG C (preferably 35 DEG C), after reaction terminates, obtain the mixed solution containing 1-cyanocyclohexanoic guanidine-acetic acid, by mixed solution separation and purification, obtain 1-cyanocyclohexanoic guanidine-acetic acid, the described engineering bacteria containing restructuring nitrilase gene gene constructedly to be formed by nucleotide sequence coded shown in SEQ ID NO.2.Described initial substrate concentration is 0.02 ~ 1mol/L, preferred 100mmol/L, and the quality final concentration of described wet thallus is 10 ~ 100g/L, preferred 50g/L.
Further, catalyzer of the present invention is prepared as follows: will be seeded in the LB liquid nutrient medium containing final concentration 50mg/L kantlex containing the engineering bacteria of nitrilase gene, 37 DEG C, 150rpm cultivates 10 ~ 12 hours, obtains seed liquor; With being seeded in the LB liquid nutrient medium containing final concentration 50mg/L kantlex by seed liquor with the inoculum size of volumetric concentration 2%, 37 DEG C, 150rpm is cultured to the OD of nutrient solution
600be 0.6 ~ 0.8, in nutrient solution, add the IPTG that final concentration is 0.1mM, 28 DEG C, 150rpm inducing culture 10 hours, by medium centrifugal, get precipitation washing, obtain wet thallus and be catalyzer; Described LB liquid nutrient medium consists of: 10g/L Tryptones, 5g/L yeast extract, 10g/L sodium-chlor, and solvent is water, and pH value is 7.
The method of 1-cyanocyclohexanoic guanidine-acetic acid of the present invention separation and purification is generally: transform after terminating, add 7 ~ 8mL in conversion fluid, the NaOH solution of 10.5M (makes product comparatively good dissolving, improve the rate of recovery), adjust pH to 8.5-9.0, centrifugal (9000rpm, 10min), supernatant liquor is got.In supernatant liquor, 1:1 ratio adds aqueous ethanolic solution (water content 5% volumetric concentration) (removing the nucleic acid that bacterial cell disruption produces, protein etc.) by volume, suction filtration.Get filtrate, underpressure distillation (temperature controls below 50 DEG C).Distillate in the remaining liq after alcohol the HC-767 injection-use activated carbon adsorbing contaminant adding 5 ‰, suction filtration.Add hydrochloric acid in filtrate and adjust about pH to 2.5, leave standstill, suction filtration, the filtrate recovery containing single acid in funnel is dried (less than 35 DEG C), namely obtain product 1-cyanocyclohexanoic guanidine-acetic acid.
The reactional equation that biocatalysis 1-cyanocyclohexanoic base acetonitrile of the present invention prepares 1-cyanocyclohexanoic guanidine-acetic acid is:
The construction process of the engineering bacteria containing nitrilase gene of the present invention is: utilize protein (PDB) and (NCBI) database to screen nitrilase gene sequence, based on the nitrilase gene of prunosus red coccus K22 (Rhodococcus rhodochrous K22) (GeneBank accession no.D12583) sequence, codon according to intestinal bacteria preference carries out codon optimized, the nitrilase gene RkN shown in SEQ ID NO.2 has been synthesized with complete synthesis method by engineered routine operation, the aminoacid sequence of the nitrilase of this genes encoding is for shown in SEQ ID NO.1.Then above-mentioned nitrilase gene is proceeded in carrier and build recombinant expression vector pET28b (+) – RkN, transform Host Strains (preferred e. coli bl21 (DE3)) with recombinant expression vector again, build the engineering bacteria containing nitrilase gene.
Through sequence alignment, Rhodococcus rhodochrous K22 nitrilase is respectively 68.15% and 40.31% with nitrilase bll6402 (the GeneBank accession no.BAC51667) homology having been reported the catalysis activity of nitrilase-Acidovorax facilis 72W nitrilase (GeneBank accession no.ABD98457) and Bradyrhizobium japonicum USDA110 have to(for) 1-cyanocyclohexanoic base acetonitrile.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the invention provides a kind of method that microorganism catalysis 1-cyanocyclohexanoic base acetonitrile prepares 1-cyanocyclohexanoic guanidine-acetic acid, engineering bacteria containing nitrilase of the present invention is as catalyzer, have good regioselectivity and very high catalysis activity (live than enzyme and reach 4800U/g), production process is environmentally friendly; Solve that traditional chemical method hydrolysis 1-cyanocyclohexanoic base acetonitrile is a large amount of organic solvent of severe reaction conditions in the technique of 1-cyanocyclohexanoic guanidine-acetic acid, reaction needed, cost is higher, yield is lower, the problem that environmental pollution is comparatively serious.
(4) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Following examples major experimental material source used is:
E. coli host bacteria strain E.coli BL21 (DE3) is purchased from Invitrogen company, expression vector pET-28b (+) is purchased from Novagen company, restriction enzyme Nco I and Xho I is purchased from Fermentas company, and T4 DNA ligase and kantlex are all purchased from the precious biological company limited in Dalian; Isopropyl-beta D-thio galactopyranoside (IPTG) is Promega Products.DNA Marker and staining agent Gold View is purchased from TaKaRa company; Axygen DNA gel reclaims test kit, plasmid extraction test kit, PCR purification kit purchased from liking to pursue progress Bioisystech Co., Ltd.
Embodiment 1: the synthesis of nitrilase gene
Utilize protein PDB and ncbi database to screen nitrilase gene sequence, obtain a nitrilase gene (GeneBank accession no.D12583).This nitrilase derives from prunosus red coccus K22 (Rhodococcus rhodochrous K22), according to the sequence (GeneBank accession no.D12583) of Rhodococcus rhodochrous K22 nitrilase, codon according to intestinal bacteria preference carries out codon optimized, restriction enzyme site Nco I is devised according to the feature of expression vector pET28b (+), Xho I, this section of nitrilase gene RkN (shown in SEQ ID NO.2) has been synthesized with complete synthesis method by engineered routine operation, the aminoacid sequence of codase is for shown in SEQ ID NO.1.
Embodiment 2: the structure of nitrilase gene expression vector and recombinant conversion thereof
Nitrilase gene RkN and the pET28b (+) of synthesis carries out double digestion with Nco I and Xho I respectively, after about enzyme cuts 6h, reclaim digestion products, utilize T4 ligase enzyme to connect 16 hours at 16 DEG C, obtain recombinant expression plasmid pET28b (+)-RkN.Expression vector pET28b (+)-RkN is converted into E.coli BL21 (DE3) recipient bacterium, coat on the LB agar plate containing kantlex (final concentration 50mg/L), after 37 DEG C of overnight incubation, flat board grows bacterium colony (yellow-white circular colonies).Random picking mono-clonal, extract plasmid after cultivating and check order, sequencing result shows to obtain positive colony E.coli BL21 (DE3)/pET28b (+)-RkN.
Embodiment 3: the expression of nitrilase
Sub-E.coli BL21 (DE3)/pET28b (+)-RkN of the recombinant conversion obtained in embodiment 2 is inoculated in LB liquid nutrient medium, 37 DEG C, 150rpm cultivates 10-12 hour, then the LB liquid nutrient medium that by volume inoculum size of concentration 2% is seeded to containing kantlex (final concentration 50mg/L) carries out enlarged culturing, 37 DEG C, 150rpm is cultured to the OD of nutrient solution
600between 0.6-0.8, adding isopropyl-beta D-thio galactopyranoside (IPTG) to final concentration is 0.1mM, 28 DEG C, 150rpm inducing culture 10 hours.Medium centrifugal collects thalline, cleans 2 times obtain wet thallus with physiological saline.
Embodiment 4: nitrilase vigor is verified
Resting cell catalyzed reaction is carried out to the wet thallus obtained in embodiment 3, to verify the vigor of nitrilase for 1-cyanocyclohexanoic base acetonitrile.
Experimental technique: take 0.5g wet thallus (final concentration 50g/L) and be suspended in (pH=7.0 in 10mL Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution system, 0.2M), add 0.296g1-cyanocyclohexanoic base acetonitrile (final concentration 200mM), 35 DEG C of water bath with thermostatic control reaction 30min.After having reacted, get 1mL reaction solution, 12000rpm is centrifugal, discards precipitation, gets supernatant liquor high performance liquid chromatography (C18 post) assay products concentration.Efficient liquid phase chromatographic analysis condition is: moving phase is damping fluid (0.58g primary ammonium phosphate+1.83g sodium perchlorate is dissolved in 1L pure water, regulates pH to be 1.8 with perchloric acid): acetonitrile=76: 24, column temperature is 40 DEG C.Under similarity condition, contrast with E.coli BL21 (DE3) empty carrier and E.coli BL21 (DE3)/pET28b (+)-RkN do not induced and E.coli BL21 (DE3)/pET28b (+)-AcN containing Acidovorax facilis 72W nitrilase, result is as shown in table 1.Enzyme is lived and is defined as: under certain condition, the per minute catalytic substrate enzyme amount of producing needed for 1 μm of ol product is defined as a unit of activity, is designated as U.
Table 1: nitrilase RkN vigor the result
Embodiment 5: use the recombination bacillus coli containing nitrilase RkN to transform 100mM 1-cyanocyclohexanoic base acetonitrile.
Experimental technique: (prepared by embodiment 3 to take 0.5g wet thallus, final concentration 50g/L) be suspended in (pH=7.0 in 10mL Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution system, 0.2M), add 0.148g1-cyanocyclohexanoic base acetonitrile (final concentration 100mM), 35 DEG C of waters bath with thermostatic control react 4 hours.After having reacted, get 1mL reaction solution, 12000rpm is centrifugal, discards precipitation, gets supernatant liquor high performance liquid chromatography (C18 post) assay products concentration.Efficient liquid phase chromatographic analysis condition is: moving phase is damping fluid (0.58g primary ammonium phosphate+1.83g sodium perchlorate is dissolved in 1L pure water, regulates pH to be 1.8 with perchloric acid): acetonitrile=76: 24, column temperature is 40 DEG C.Result: the concentration of product 1-cyanocyclohexanoic guanidine-acetic acid is 99mM, and productive rate is 99%.
Embodiment 6: use the recombination bacillus coli containing nitrilase RkN to transform 200mM 1-cyanocyclohexanoic base acetonitrile
Experimental technique: (prepared by embodiment 3 to take 0.5g wet thallus, final concentration 50g/L) be suspended in (pH=7.0 in 10mL Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution system, 0.2M), add 0.296g1-cyanocyclohexanoic base acetonitrile (final concentration 200mM), 35 DEG C of water bath with thermostatic control reaction 30min.After having reacted, get 1mL reaction solution, 12000rpm is centrifugal, discards precipitation, gets supernatant liquor high performance liquid chromatography (C18 post) assay products concentration.Efficient liquid phase chromatographic analysis condition is: moving phase is damping fluid (0.58g primary ammonium phosphate+1.83g sodium perchlorate is dissolved in 1L pure water, regulates pH to be 1.8 with perchloric acid): acetonitrile=76: 24, column temperature is 40 DEG C.
Result is as follows: the concentration of product 1-cyanocyclohexanoic guanidine-acetic acid is 192mM, and productive rate is 96%.
Embodiment 7: use the recombination bacillus coli containing nitrilase RkN to transform 500mM 1-cyanocyclohexanoic base acetonitrile
Experimental technique: take 10g wet thallus (final concentration 100g/L) prepared by embodiment 3 method and be suspended in (pH=7.0 in 100mL Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution system, 0.2M), add 7.4g1-cyanocyclohexanoic base acetonitrile (final concentration 500mM), 35 DEG C of waters bath with thermostatic control react 12 hours.Reaction solution uses the extraction into ethyl acetate of 2 times of volumes, discards organic phase.Aqueous phase adds acidifying with acetic acid, then adds the extraction into ethyl acetate of 2 times of volumes.Organic phase 35 DEG C oven dry, obtains 1-cyanocyclohexanoic guanidine-acetic acid.
Result is as follows: obtain product 1-cyanocyclohexanoic guanidine-acetic acid 6.75g, productive rate is 91.2%.
Claims (5)
1. the method utilizing nitrilase engineering bacteria to prepare 1-cyanocyclohexanoic guanidine-acetic acid, it is characterized in that described method is: by the medium centrifugal of engineering bacteria after fermentation culture containing restructuring nitrilase gene, using wet thallus as catalyzer, with 1-cyanocyclohexanoic base acetonitrile for substrate, take pH value as the damping fluid of 3.0 ~ 10.0 be reaction medium, conversion reaction is carried out at 25 ~ 70 DEG C, after reaction terminates, obtain the mixed solution containing 1-cyanocyclohexanoic guanidine-acetic acid, by mixed solution separation and purification, obtain 1-cyanocyclohexanoic guanidine-acetic acid; The described engineering bacteria containing restructuring nitrilase gene gene constructedly to be formed by nucleotide sequence coded shown in SEQ ID NO.2.
2. utilize nitrilase engineering bacteria to prepare the method for 1-cyanocyclohexanoic guanidine-acetic acid as claimed in claim 1, it is characterized in that described initial substrate concentration is 0.02 ~ 1mol/L, the quality final concentration of described wet thallus is 10 ~ 100g/L.
3. utilize nitrilase engineering bacteria to prepare the method for 1-cyanocyclohexanoic guanidine-acetic acid as claimed in claim 1, it is characterized in that described catalyzer is prepared as follows: be seeded in the LB liquid nutrient medium containing final concentration 50mg/L kantlex by the engineering bacteria containing nitrilase gene, cultivate 10 ~ 12 hours for 37 DEG C, obtain seed liquor; With being seeded in the LB liquid nutrient medium containing final concentration 50mg/L kantlex by seed liquor with the inoculum size of volumetric concentration 2%, 37 DEG C of OD being cultured to nutrient solution
600be 0.6 ~ 0.8, in nutrient solution, add the IPTG that final concentration is 0.1mM, 28 DEG C of inducing culture 10 hours, by medium centrifugal, get precipitation washing, obtain wet thallus and be catalyzer; Described LB liquid nutrient medium consists of: 10g/L Tryptones, 5g/L yeast extract, 10g/L sodium-chlor, and solvent is water, and pH value is 7.
4. utilize nitrilase engineering bacteria to prepare the method for 1-cyanocyclohexanoic guanidine-acetic acid as claimed in claim 1, it is characterized in that described conversion reaction medium to be pH value be the phosphoric acid buffer of 7.
5. utilize nitrilase engineering bacteria to prepare the method for 1-cyanocyclohexanoic guanidine-acetic acid as claimed in claim 1, it is characterized in that described conversion reaction temperature is 35 DEG C.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962540A (en) * | 2015-06-30 | 2015-10-07 | 浙江工业大学 | Nitrilase, encoding genes, carrier and application |
| CN105602922A (en) * | 2015-10-30 | 2016-05-25 | 浙江工业大学 | Pantoea amidase, gene, vector, engineering bacterium and application thereof |
| CN111100856A (en) * | 2020-01-13 | 2020-05-05 | 浙江工业大学 | Nitrilase mutant and application thereof in synthesis of pregabalin chiral intermediate |
| CN112852790A (en) * | 2018-07-12 | 2021-05-28 | 浙江工业大学 | Plant nitrilase chimeric enzyme mutant, coding gene and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050009157A1 (en) * | 2000-03-30 | 2005-01-13 | Canon Kabushiki Kaisha | Polyhydroxyalkanoate synthase and gene encoding the same enzyme |
| CN101629192A (en) * | 2009-07-16 | 2010-01-20 | 浙江工业大学 | Method for preparing iminodiacetic acid by catalyzing iminodiacetonitrile with microbes |
| CN101918573A (en) * | 2007-10-31 | 2010-12-15 | 纳幕尔杜邦公司 | Sequestration of formaldehyde to stabilize nitrilase specific activity when converting glycolonitrile to glycolic acid |
| CN102911975A (en) * | 2012-09-12 | 2013-02-06 | 浙江工业大学 | Method for preparing 2-amino-4-methylthio butyric acid by using recombinant nitrilase |
-
2014
- 2014-08-12 CN CN201410395753.7A patent/CN104212850B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050009157A1 (en) * | 2000-03-30 | 2005-01-13 | Canon Kabushiki Kaisha | Polyhydroxyalkanoate synthase and gene encoding the same enzyme |
| CN101918573A (en) * | 2007-10-31 | 2010-12-15 | 纳幕尔杜邦公司 | Sequestration of formaldehyde to stabilize nitrilase specific activity when converting glycolonitrile to glycolic acid |
| CN101629192A (en) * | 2009-07-16 | 2010-01-20 | 浙江工业大学 | Method for preparing iminodiacetic acid by catalyzing iminodiacetonitrile with microbes |
| CN102911975A (en) * | 2012-09-12 | 2013-02-06 | 浙江工业大学 | Method for preparing 2-amino-4-methylthio butyric acid by using recombinant nitrilase |
Non-Patent Citations (1)
| Title |
|---|
| 徐赛珍等: "选择性腈水解酶在生物催化中的应用", 《浙江化工》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962540A (en) * | 2015-06-30 | 2015-10-07 | 浙江工业大学 | Nitrilase, encoding genes, carrier and application |
| CN105602922A (en) * | 2015-10-30 | 2016-05-25 | 浙江工业大学 | Pantoea amidase, gene, vector, engineering bacterium and application thereof |
| CN105602922B (en) * | 2015-10-30 | 2019-05-28 | 浙江工业大学 | The general raw bacterium amidase of one kind, gene, carrier, engineering bacteria and its application |
| CN112852790A (en) * | 2018-07-12 | 2021-05-28 | 浙江工业大学 | Plant nitrilase chimeric enzyme mutant, coding gene and application thereof |
| CN111100856A (en) * | 2020-01-13 | 2020-05-05 | 浙江工业大学 | Nitrilase mutant and application thereof in synthesis of pregabalin chiral intermediate |
| CN111100856B (en) * | 2020-01-13 | 2021-12-07 | 浙江工业大学 | Nitrilase mutant and application thereof in synthesis of pregabalin chiral intermediate |
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